Cryptosporidium spp are protozoan parasites pathogenic to humans listed as Class B Priority pathogens. In spite of what is known about its biology, several questions concerning the dynamics of transmission and identification of infection sources have been proven difficult to investigate using microscopy: 1) What is the public health significance of Cryptosporidium spp identified in animals? 2) What is the disease burden attributable to parasites of animal origin? 3) Is there a temporal or geographic clustering of Cryptosporidium spp or subtypes? Recently molecular tools have been developed to allow for the species differentiation and sub-typing of Cryptosporidium and could be used to address these questions. This project will apply molecular tools and Real-Time PCR technology to detect and type Cryptosporidium in stool specimens and household animals. Positive samples will be further sub-typed by DNA sequencing. Samples will be collected from 350 children ages 10 year or younger who are already enrolled in an on-going TMRC study where they provide weekly stool samples and epidemiological risk factor information including socioeconomic and symptoms data. This project will add: a) the collection and of stool samples from all animals and family members living in the households of positive children; b) microscopy and molecular testing for Cryptosporidium spp. for all samples; c) establishment of a case-control study using two negative households for each Cryptosporidium-positive child, and d) GIS mapping of Cryptosporidium positive cases. DNA will be extracted from all microscopy-positive samples and the species of Cryptosporidium will be determined by Real-Time PCR and melting curve analysis (PCR-MCA). To determine the sub-types of Cryptosporidium and the robustness of the PCR-MCA, all isolates of C. hominis, C. parvum bovine genotype and C. meleagridis will be sequenced at the GP-60 gene locus. Data from the case-control study and GIS mapping will be analyzed for risk factor associations and geographic distribution of cases and subtypes within the community. The information generated through this project will enhance the understanding of the sources of infections the transmission of cryptosporidiosis, the distribution of Cryptosporidium genotypes in animals, sources of infection, and clustering of sub-types.